Identification and expression profile of odorant-binding proteins in the parasitic wasp Microplitis pallidipes using PacBio long-read sequencing

Abstract

Microplitis pallidipes Szépligeti (Hymenoptera: Braconidae) is an important parasitic wasp of second and third-instar noctuid larvae such as the insect pests Spodoptera exigua, Spodoptera litura, and Spodoptera frugiperda. As in other insects, M. pallidipes has a chemosensory recognition system that is critical to foraging, mating, oviposition, and other behaviors. Odorant-binding proteins (OBPs) are important to the system, but those of M. pallidipes have not been determined. This study used PacBio long-read sequencing to identify 170,980 M. pallidipes unigenes and predicted 129,381 proteins. Following retrieval of possible OBP sequences, we removed those that were redundant or non-full-length and eventually cloned five OBP sequences: MpOBP2, MpOBP3, MpOBP8, MpOBP10, and MpPBP 429, 429, 459, 420, and 429 bp in size, respectively. Each M. pallidipes OBP had six conserved cysteine residues. Phylogenetic analysis revealed that the five OBPs were located at different branches of the phylogenetic tree. Additionally, tissue expression profiles indicated that MpOBP2 and MpPBP were mainly expressed in the antennae of male wasps, while MpOBP3, MpOBP8, and MpOBP10 were mainly expressed in the antennae of female wasps. MpOBP3 was also highly expressed in the legs of female wasps. Temporal profiles revealed that the expression of each M. pallidipes OBP peaked at different days after emergence to adulthood. In conclusion, we identified five novel odorant-binding proteins of M. pallidipes and demonstrated biologically relevant differences in expression patterns.

Keywords: Biological control; Odorant-binding protein; Parasitic insect; Tissue and temporal expression profile; Transcriptome sequencing.

Figure 1

Overview of PacBio sequencing in Microplitis pallidipes. (a) Numbers of different types of PacBio reads in M. pallidipes. Blue and green circles represent 3′ and 5′ primer reads, respectively while green indicates their intersection. The red circle represents the full-length non-concatemer reads. (b) Length distribution of M. pallidipes unigenes obtained using PacBio Iso-Seq. Gene length is on the x-axis and number of genes is on the y-axis.

Figure 2

Annotation of transcripts using public databases. (a) Homologous species distribution of M. pallidipes annotated using the NR database. (b) GO enrichment analysis: red, biological process; green, cellular component; blue, molecular function. (c) KEGG pathway analysis: red, metabolism; green, genetic information processing; blue, environmental information processing; yellow, cellular processes; purple, organismal systems. (d) eggNOG annotations: eggNOG categories are on the x-axis and number of transcripts is on the y-axis.

Figure 3

Analysis and prediction of simple sequence repeats, open reading frames, and transcription factors. (a) Simple sequence repeats of M. pallidipes transcripts. Number of bases repeated is on the x-axis and amount of sequence repeats is on the y-axis. (b) Analysis of open reading frames (ORF). Red, ORFs annotated by NR and SwissProt; blue, ORFs predicted using Transdecoder. (c) Transcription factors predicted using SMRT. Transcription-factor family is on the x-axis and number of transcription factors is on the y-axis.

Figure 4

Sequence characteristics analysis of five M. pallidipes odorant-binding proteins (OBPs). Green represents signal peptides, blue represents conserved sequences of the PBP/GOBP family, and red represents cysteine. Numbers indicate amino acid position.

Figure 5

Phylogenetic tree of OBP amino acid sequences reconstructed in MEGA7. Branch lengths indicate evolutionary distances, and numbers represent the tree confidence calculated using bootstrap analysis with 1000 replicates. Different colors of text represent OBPs of different species: red, Microplitis pallidipes (Mp); green, Apis mellifera (Am); blue, Microplitis mediator (Mm); yellow, Dichasma alloeum (Da).

Figure 6

Relative quantification of M. pallidipes OBPs in different tissues of female and male wasps. (a–e) MpOBP2, MpOBP3, MpOBP8, MpOBP10, and MpPBP, respectively. The two charts on the left represent comparisons of OBP expression in different tissues, and the six small charts on the right represent comparisons of female and male OBP expression. Tissue type or sex is on the x-axis and relative quantification of M. pallidipes OBPs is on the y-axis. Bars represent standard deviations. An: antennae, H: heads, T: thoraxes, Ab: abdomens, W: wings, L: legs; F: female, M: male. Different lower-case letters indicate a significant difference (one-way ANOVA, p < 0.05). ns means no significant difference and * means a significant difference (t-test, p < 0.05).

Figure 7

Relative quantification of M. pallidipes OBPs at different days after emergence to adulthood in female and male wasps. (a–e) MpOBP2, MpOBP3, MpOBP8, MpOBP10, and MpPBP, respectively. The two charts on the left represent comparisons of OBP expression at different days after emergence to adulthood, and the five small charts on the right represent comparisons of female and male OBP expression. Day or sex is on the x-axis and relative quantification of M. pallidipes OBPs is on the y-axis. Bars represent standard deviations. 1d–5d: 1-day-old adults, 2-day-old adults, 3-day-old adults, 4-day-old adults, and 5-day-old adults; F: female, M: male. Different lower-case letters indicate a significant difference (one-way ANOVA, p < 0.05). ns means no significant difference and * means a significant difference (t-test, p < 0.05).